Differential lipidomics of HK-2 cells and exosomes under high glucose stimulation

Abstract

Abnormal cellular lipid metabolism has a very important role in the occurrence and progression of diabetic kidney disease (DKD). However, the lipid composition and differential expression by high glucose stimulation of renal tubular cells and their exosomes, which is a vital part of the development of DKD, are largely unknown. In this study, based on targeted lipid analysis by isotope labeling and tandem mass spectrometry, a total of 421 and 218 lipid species were quantified in HK-2 cells and exosomes, respectively. More importantly, results showed that GM3 d18:1/22:0, GM3 d18:1/16:0, GM3 d18:0/16:0, GM3 d18:1/22:1 were significantly increased, while LPE18:1, LPE, CL66:4 (16:1), BMP36:3, CL70:7 (16:1), CL74:8 (16:1) were significantly decreased in high glucose-stimulated HK-2 cells. Also, PG36:1, FFA22:5, PC38:3, SM d18:1/16:1, CE-16:1, CE-18:3, CE-20:5, and CE-22:6 were significantly increased, while GM3 d18:1/24:1, GM3 were significantly decreased in exosomes secreted by high glucose-stimulated HK-2 cells. Furthermore, TAG, PC, CL were decreased significantly in the exosomes comparing with the HK-2 cells, and LPA18:2, LPI22:5, PG32:2, FFA16:1, GM3 d18:1/18:1, GM3 d18:1/20:1, GM3 d18:0/20:0, PC40:6p, TAG52:1(18:1), TAG52:0(18:0), CE-20:5, CE-20:4, CE-22:6 were only found in exosomes. In addition, the expression of PI4P in HK-2 cells decreased under a high glucose state. These data may be useful to provide new targets for exploring the mechanisms of DKD.

Keywords: Diabetic kidney disease; Exosomes; Renal tubules; Targeted lipidomics.

Fig 1

HK-2 cells lipid composition and differential expression under high glucose stimulation. A Species detected in each 21 major lipids of HK-2 cell. B Average lipid content of each 21 major lipids in HK-2 cells. C The expression of GM3 d18:1/22:0, GM3 d18:1/16:0, GM3 d18:0/16:0, GM3 d18:1/22:1 were significantly increased in HG, while the expression of LPE18:1, LPE, CL66:4(16:1), BMP36:3, CL70:7(16:1), CL74:8(16:1) were significantly decreased in HG. D The heatmap of GM3. The expression of GM3 d18:1/22:0, GM3 d18:1/16:0, GM3 d18:0/16:0, and GM3 d18:1/22:1 were all significantly increased in HG. E The heatmap of CL. The expression of CL66:4(16:1), CL70:7(16:1), and CL74:8(16:1) were all significantly decreased in HG. CL, cardiolipin; GM3, monosialodihexosyl ganglioside; NG, control group; HG, high glucose group.

Fig 2

Exosomes lipid composition and differential expression under high glucose stimulation. A Representative electron micrograph images of exosomes secreted by HK-2 cells. Scale bar, 100nm. B, C Size distribution of exosomes secreted by HK-2 cells using NTA. D Western blotting of exosomal markers (including Alix and CD63) and endoplasmic reticulum molecular chaperon Calnexind in exosomes secreted by HK-2 cells, cells used as control. E Species detected in each 21 major lipids of exosomes secreted by HK-2 cells. F Average lipid content of each 21 major lipids in exosomes secreted by HK-2 cells. G In exosomes secreted by HK-2 cells, the expression of PG36:1, FFA22:5, PC38:3, SM d18:1/16:1, CE-16:1, CE-18:3, CE-20:5, CE-22:6 were significantly increased in HG, while the expression of GM3 d18:1/24:1, GM3 were significantly decreased in HG. H CE-20:5, CE-16:1, CE-18:3, GM3 d18:1/24:1 were the first four metabolites with the most significant differences between HG and NG groups. NG, control group; HG, high glucose group; NG-Exo, exosomes secreted by NG; HG-Exo, exosomes secreted by HG.

Fig 3

Differential expression of exosomes, autophagy and PIPs in HK-2 cells under high glucose stimulation. A Changes in volume of cellular MVBs in HK-2 cells after high glucose stimulation for 48h by transmission electron microscopy (the arrows show the MVBs). B Changes of the amount of exosomes protein secreted by HK-2 cells after high glucose stimulation for 48h by BCA analysis. C Changes of the expression of LC3 and P62 in HK-2 cells after high glucose stimulation by western blotting. D Differential expression of PIPs in HK-2 cells after high glucose stimulation for 48h. E The heatmap of PIPs. PIPs, Phosphoinositides; NG, control group; HG, high glucose group; NG-Exo, exosomes secreted by NG; HG-Exo, exosomes secreted by HG. ^*P<0.05, ^***P<0.001.

Fig 4

Uptake of HK-2 cells-derived exosomes by GMCs. HK-2 cells-derived exosomes were fluorescently labeled with PKH67 (green), then incubated with GMCs. Cell nuclei were stained with DAPI (blue). A confocal microscope was used to visualize the uptake of labeled exosomes by GMCs. GMCs, glomerular mesangial cells; NG, control group; HG, high glucose group; NG-Exo, exosomes secreted by NG; HG-Exo, exosomes secreted by HG.